Monoclonal antibodies against human apoptosis inhibitory protein NAIP and method for assaying the NAIP

ABSTRACT

An anti-NAIP monoclonal antibody recognizing a human apoptosis inhibitory protein NAIP having the amino acid sequence of SEQ ID NO: 1, which is produced from hybridoma prepared by fusing a myeloma cell line with antibody-producing cell of mammal immunized by antigen containing a polypeptide which comprises amino acid sequence of the 256-586th, the 841-1052nd or parts thereof in SEQ ID NO: 1, NAIP assay method using the antibody, and NAIP assay kits.

TECHNICAL FIELD

[0001] The present invention relates to monoclonal antibodies, whichspecifically recognize Human Apoptosis Inhibitory Protein NAIP and animmunoassay method of the NAIP.

BACKGROUND ART

[0002] Apoptosis is a type of programmed death of a cell, in whichphenomena such as lack of contact with the surrounding cells,inspissation of cytoplasm, aggregation of chromatin and karyopyknosisrelated to the activity of endonuclease, fragmentation of nucleus, thecell being changed into membrane-wrapped bulboid corpuscles, englobementof the bulboid corpuscles by the adjacent macrophage or epithelialcells, or fragmentation of the nucleosome unit of DNA into DNA fragmentsof 180-200 base length by the activity of endonuclease are observed.Apoptosis has been discussed as a mechanism in which the final fragmentsof apoptic somatic cells exhibiting the aforementioned phenomena areenglobed by the adjacent cells (e.g., “Immunology Today”, 7:115-119.1986: Science 245:301-305. 1989).

[0003] As the gene that controls the apoptosis described above, thebcl-2 gene, which is one of oncogene discovered from B cell lymphoma in1985, is known. This bcl-2 gene appears quite frequently in cells of theimmune system or neuronal cells. It is assumed that the substanceproduced as a result of expression of the gene suppresses the apoptosisof such cells, whereby the function of the human immune and neuronalsystems are constantly maintained the homeostasis thereof. In addition,as the bcl-2 gene appears in a fetus in an especially wide range, it isassumed that the gene plays an important role in morphogeny during theontogenic process.

[0004] On the other hand, the inventors of the present application haveisolated Neuronal Apoptosis Inhibitory Protein (NAIP) gene, as the genecausing Spinal Muscular Atrophy (SMA) which is a familial geneticdisease, from the human chromosome 5q13.1 domain (Roy et al., Cell 80:167-178, 1995), and they have filed a patent application for the gene(PCT/CA95/00581). Specifically, it is considered that the mutation ofthe NAIP gene or the decrease in the number of copies therefrom causesapoptosis of spinal neurons, resulting in the development of SMA.Further, in a case in which the NAIP gene is introduced into variouscultured cells and stimulation is provided to the cells to induceapoptosis, it has been found out that the death of the cells issignificantly prevented (Liston et al., “Nature” 379: 349-353, 1996). Inthis case, it has also been found out that NAIP functions as theapoptosis inhibitory factor not only to the neuronal cells but also tothe somatic cells as a whole.

[0005] The inventors of the present application have isolated the fullamino acid sequence of NAIP and cDNA encoding the NAIP, and filed apatent application thereof (Japanese Patent Application No. 9-280831).

[0006] As described above, NAIP is a protein which is concerned withvarious apoptosis-related diseases including SMA. In order to understandthe mechanism of a patient's developing such diseases, diagnose the riskfor developing the diseases, prevent the development of the diseases orreduce the severity of the diseases, and develop the medical techniqueand medicines for treatment, it is essential to accurately assay theamount of expressed NAIP.

[0007] The inventions of the present application has been contrived inconsideration of the aforementioned task, and objects of the presentinvention is to provide anti-NAIP monoclonal antibodies, which areessential for assaying NAIP and a NAIP assaying method using themonoclonal antibodies.

DISCLOSURE OF INVENTION

[0008] The inventors of the present application have assiduously studiedthe solution of the aforementioned task, and as a result, discoveredthat the epitopes of NAIP exist in the amino acids of the 256-586th andthe 841-1052nd in SEQ ID NO: 1.

[0009] The present application, on the basis of the discovery, providesan anti-NAIP monoclonal antibody recognizing a human apoptosisinhibitory protein NAIP having the amino acid sequence of SEQ ID NO: 1,which is produced from hybridoma prepared by fusing a myeloma cell linewith antibody-producing cell of mammal immunized by antigen containing apolypeptide which comprises amino acid sequence of the 256-586th, the841-1052nd or parts thereof in SEQ ID NO:1

[0010] The present application provides, as specific embodiments of themonoclonal antibody: anti-NAIP monoclonal antibody hnmc365, which isproduced from hybridoma 656-1 (FERM BP-6919), and its epitope is the354-365th region in SEQ ID NO: 1.; anti-NAIP monoclonal antibodyhnmc381, which is produced from hybridoma 656-2 (FERM BP-6920), and itsepitope is the 373-387th region in SEQ ID NO: 1; and anti-NAIPmonoclonal antibody hnmc841, which is produced from hybridoma 841 (FERMBP-6921), and its epitope is the 841-1052nd region in SEQ ID NO:1.

[0011] The present application provides the first method of assayingNAIP, which comprises contacting a marker-labeled anti-NAIP monoclonalantibody with a sample containing NAIP thereby binding themarker-labeled antibody with NAIP, and measuring signal strength of themarker in the bound structure.

[0012] In the first assay method, it is preferred that the anti-NAIPmonoclonal antibody is any one of said hnmc365, hmnc381 and hmnc841, andthat the marker is an enzyme, a radioactive isotope or a fluorescentcolorant.

[0013] The present application provides the second method of assayingNAIP which comprises contacting an anti-NAIP primary antibody with asample containing NAIP thereby binding the primary antibody with NAIP,further binding the bound structure with an anti-NAIP secondaryantibody, and measuring signal strength of a marker bound with thesecondary antibody, wherein:

[0014] (1) the primary antibody and the secondary antibody are both saidanti-NAIP monoclonal antibody;

[0015] (2) the primary antibody is said anti-NAIP monoclonal antibodyand the secondary antibody is an anti-NAIP polyclonal antibody; or

[0016] (3) the primary antibody is an anti-NAIP polyclonal antibody andthe secondary antibody is said anti-NAIP monoclonal antibody.

[0017] In the second assay method, it is preferred that the primaryantibody is immobilized on solid phase, that the anti-NAIP monoclonalantibody is any one of said hnmc365, hmnc381 and hmnc841, and that themarker is an enzyme, a radioactive isotope or a fluorescent colorant.

[0018] The present application provides the first kit for assaying NAIPat least including:

[0019] (a) a plate on which an anti-NAIP primary antibody isimmobilized; and

[0020] (b) an anti-NAIP secondary antibody labeled with a marker,wherein:

[0021] (1) the primary antibody and the secondary antibody are both saidanti-NAIP monoclonal antibody;

[0022] (2) the primary antibody is said anti-NAIP monoclonal antibodyand the secondary antibody is an anti-NAIP polyclonal antibody; or

[0023] (3) the primary antibody is an anti-NAIP polyclonal antibody andthe secondary antibody is said anti-NAIP monoclonal antibody.

[0024] In the first assay kit, it is preferred that the marker is aradioactive isotope, a fluorescent colorant or an enzyme, and in thecase of the marker being emzyme the kit further includes:

[0025] (c) a substrate which develops a color by the enzyme activity.

[0026] The present invention provides the second kit for assaying NAIPat least including:

[0027] (a) a plate on which an anti-NAIP primary antibody isimmobilized;

[0028] (b) an anti-NAIP secondary antibody; and

[0029] (c) a marker to be bound with the secondary antibody, wherein:

[0030] (1) the primary antibody and the secondary antibody are both saidanti-NAIP monoclonal antibody;

[0031] (2) the primary antibody is said anti-NAIP monoclonal antibodyand the secondary antibody is an anti-NAIP polyclonal antibody; or

[0032] (3) the primary antibody is an anti-NAIP polyclonal antibody andthe secondary antibody is said anti-NAIP monoclonal antibody.

[0033] In the second assay kit, it is preferred that the marker is aradioactive isotope, a fluorescent colorant or an enzyme, and in thecase of the marker being emzyme the kit further includes:

[0034] (c) a substrate which develops a color by the enzyme activity.

[0035] In said assay kits, it is further preferred that the anti-NAIPmonoclonal antibody is any one of said hnmc365, hmnc381 and hmnc841

BRIEF DESCRIPTION OF DRAWINGS

[0036]FIG. 1 is a graph that shows a relationship between theconcentration of purified NAIP in a sample solution and the absorbancemeasured by the method described in Examples.

[0037]FIG. 2 shows the results of Western Blotting in which anti-NAIPantibodies of a mononuclear cell solution derived from human peripheralblood were used. Lanes represent, in order, 1. monoclonal antibodyhnmc365; 2. monoclonal antibody hnmc381; 3. monoclonal antibody hnmc841;and 4. polyclonal antibody. The concentrations of the antibodies eachresulted from dilution by 250 times.

BEST MODE FOR CARRYING OUT THE INVENTION

[0038] The anti-NAIP monoclonal antibodies of the present invention maybe produced by the following steps, for example, according to the knownmethod (“Monoclonal Antibody” Takaaki NAGAMUNE and Hiroshi TERADA,Hirokawa Shoten, 1990; “Monoclonal Antibody” James W. Goding, thirdedition, Academic Press, 1996).

[0039] 1. Preparation of Hybridomas

[0040] A mammal animal is immunized by using an immunogen containing apolypeptide, the polypeptide comprising amino acid sequence of the256-586th, the 841-1052nd or parts thereof in SEQ ID NO: 1. Anadditional immunization is optionally carried out according to necessityso that the animal is sufficiently immunized. Next, theantibody-producing cells (lymphatic cells or spleen cells) are isolatedfrom the animals and fused cells are obtained by fusing theantibody-producing cells and myeloma cells. A plurality of cells thatrespectively produce the targeted monoclonal antibody are selected andcultured, thereby obtaining hybridomas. The steps for the procedure willbe each described in detail hereinafter.

[0041] a) Preparation of immunogen

[0042] The polypeptide having the amino acid sequence of the 265-586thin SEQ ID NO: 1 may be prepared by, for example, cleaving NAIP cDNAhaving the nucleotide sequence of SEQ ID NO: 2 with a restriction enzymeto obtain a DNA fragment containing the nucleotide sequence of the1056-2049th, and expressing the DNA fragment in an appropriatehost-vector system. The polypeptide having the amino acid sequence ofthe 841-1052th in SEQ ID NO: 1 may be prepared by expressing a DNAfragment having the nucleotide sequence of the 2812-3447th in SEQ ID NO:2 in an appropriate host-vector system.

[0043] Alternatively, polypeptide having a partial sequence (10-20 aminoacids) of the amino acids sequence of the 256-586th or the 841-1052ndregion in SEQ ID NO: 1 may be prepared. In this case, by usingpolypeptides of different sequences, hybridomas each producingmonoclonal antibody of different epitope can be obtained.

[0044] These polypeptides may be also used in a form of a fusion proteinin which the polypeptide is fused with other proteins (e.g.,glutation-S-transferase: GST). Use of such fusion proteins is especiallypreferable in terms of facilitating and ensuring the separation processof the targeted protein from the expressed product of the host-vectorsystem and the screening process (described below) of the hybridoma.

[0045] It should be noted that the polypeptide may be that having aminoacid sequence in which at least one amino acid residue is deleted orsubstituted or added in amino acid sequence of the 256-586 or a part inSEQ ID NO: 1.

[0046] b) Immunization of animals

[0047] As the animals to be immunized, mammals used in the knownhybridoma preparation methods can be employed. Specific examples of theanimals include mice, rats, goats, sheep, cows and horses. However, interms of availability of myloma cells to be fused with the isolatedtibody-producing cells, it is preferable to use mice or rats as theanimals to be immunized. There is no particular restriction on thestrains of mice and rats actually used. In the case of mice, examples ofstrains thereof which can be used include A, AKR, BALB/c, BDP, BA, CE,C3H, 57BL, C57BR, C57L, DBA, FL, HTH, HT1, LP, NZB, NZW, RF, RIII, SJL,SWR, WB, 129. In the case of rats, examples of strains thereof that canbe used include Low, Lewis, Spraque, Daweley, ACI, BN, Fisher. Amongthem, if the suitability in being fused with the myeloma cells describedbelow is considered, the “BALB/c” strain of mice and the “Low” strain ofrats are especially preferable as the animals to be immunized. It ispreferable that the mouse or rat is 5-12 week old when it is immunized.

[0048] The immunization of the animal can be carried out bysubcutaneously or intraperitoneally dozing the polypeptide solution asan immunogen, into the animal. The dosing schedule of the antigen variesdepending on the types of the subject animal or the differences betweenthe individual animals. In general, the antigen is preferably dosedtotally 2-6 times with 1-2 weeks of the interval between doses. Theamount of the antigen to be dosed also varies depending on the types ofthe animal and the differences between the individual animals. Ingeneral, the amount of the antigen to be dosed is approximately 10-100μg/μl.

[0049] c) Fusion of cells

[0050] 1-5 days after the final immunization in the aforementioneddosing schedule, spleen cells or lymphatic cells containing theantibody-producing cells are sterilely collected from the immunizedanimal. The separation of the antibody-producing cells from the spleencells or the lymphatic cells can be carried out according to the knownmethods.

[0051] Next, the antibody-producing cells are fused with myeloma cells.There is no particular restriction on the myeloma cells to be used, andthose appropriately selected from the known cell lines may be used.However, in consideration of the convenience at the time of selectinghybridomas from the fused cells, it is preferable to employ a HGPRT(Hpoxanthine-guanine phosphoribosyl transferase) defective line forwhich a selection procedure has been established. Specific examplesthereof include: X63-Ag8(X63), NS1-Ag4/1(NS-1), P3X63-Ag8.UI(P3UI),X63-Ag8.653(X63.653), SP2/0-Ag14(SP2/0), MPC11-45.6TG1.7(45.6TG), FO,S149/5XXO.BU.1, which are derived from mice; 210.RSY3.Ag.1.2.3(Y3)derived from rats; and U266AR(SKO-007), GM1500•GTG-A12(GM1500), UC729-6,LICR-LOW-HMy2(HMy2), 8226AR/NIP4-1(NP41), which are derived from human.

[0052] The antibody-producing cells may be fused with the myeloma cellsin an appropriate manner, according to the known method, under acondition in which the survival rate of the cells does not drop to suchan extremely low level. Examples of such methods include a chemicalmethod in which the antigen-producing cells are mixed with the myelomacells in a polymer (e.g., polyethylene glycol) solution of a highconcentration, a physical method in which electric stimulation isutilized, and the like.

[0053] The selection of the fused cells from the non-fused cells ispreferably carried out according to the known HAT(Hpoxanthine/Aminopterin/Thymidine) selection method. This method iseffective when fused cells are obtained by using myeloma cells of aHGPRT defective line that is not viable under the presence ofaminopterin. That is, by cultivating fused cells and cells which havenot been fused in a HAT culture, only the fused cells that is resistantto aminopterin are selectively remained and allowed to reproduce.

[0054] d) Screening of hybridoma

[0055] The screening of the hybridoma which produce the targetedmonoclonal antibody can be performed by the known EIA (EnzymeImmunoassay), RIA (Radio Immunoassay), fluorescent antibody methods andthe like. When a fused protein is employed as the immunogen, thehybridoma can be screened more reliably by carrying out theaforementioned screening methods for the protein which is the partner ofthe fusion, as well.

[0056] By conducting such a screening process, hybridomas respectivelyproducing monoclonal antibodies having different epitope domains areobtained. Accordingly, the monoclonal antibodies of the presentinvention include all of the plural types of monoclonal antibodiesrespectively produced by the hybridomas prepared by the method describedabove.

[0057] After the screening process, the hybridomas are then subjected tocloning by the known methods such as the methylcellulose method, thesoft agarose method and the limiting dilution method, so that thehybridomas can be used for producing the antibodies.

[0058] The hybridomas obtained by the aforementioned method can bestored in the frozen state in liquid nitrogen or in a freezer in whichthe temperature is no higher than −80° C.

[0059] 2. Production of the Monoclonal Antibodies and PurificationThereof

[0060] The monoclonal antibodies that specifically recognize NAIP can beobtained by cultivating, according to the known method, the hybridomasprepared as described in the paragraph 1 above.

[0061] The cultivation may be conducted, for example, in the culturehaving the same composition as that used in the cloning method describedabove. Alternatively, in order to produce a large amount of themonoclonal antibodies, it is acceptable to inject the hybridomaintraperitoneally to a mouse and collect the monoclonal antibody fromthe ascites of the animal.

[0062] The monoclonal antibody obtained in such a manner can be purifiedby the methods including the ammonium sulfate salting out method, thegel filtration method, the ion-exchange chromatography method, theaffinity chromatography method and the like.

[0063] Next, the NAIP assay method of the present invention will bedescribed hereinafter.

[0064] In the first assay method, a solution of the marker-labeledanti-NAIP monoclonal antibody (M-mAb) is contacted with a samplecontaining NAIP so that the marker-labeled monoclonal antibody is boundwith NAIP, and the bound structure (M-mAb: NAIP) are separated. As themeans for separation, any known methods including the chromatographymethod, the salting out method, the alcohol precipitation method, theenzyme method, the solid phase method and the like may be employed. In acase in which an enzyme is used as the marker, a substrate that developsa color as a result of decomposition by the enzyme activity is added. Inthis case, the activity of the enzyme is measured by optically measuringthe amount of the decomposed substrate and the activity of the enzyme isconverted into the amount of bound antibody, so that the amount of NAIPis calculated on the basis of the comparison of the obtained amount ofbound antibody with the reference value. In a case in which aradioactive isotope is used as the marker, the amount of the radioactiverays emitted from the radioactive isotope is measured by a scintillationcounter or the like. In a case in which a fluorescent colorant is usedas the marker, a device in which a fluorescent microscope isincorporated can measure the magnitude of fluorescence.

[0065] In the second assay method, two types of antibodies whose epitopedomains for NAIP are different from each other (the primary antibody andthe secondary antibody) are used. Specifically, at first the primaryantibody (Ab I) is contacted with a sample containing NAIP so that theprimary antibody and NAIP are bound with each other. The bound structure(Ab I: NAIP) is bound with the secondary antibody that has beenmarker-labeled (M-Ab II), and the signal strength of the marker in thebound structure of the three components (Ab I: NAIP: M-Ab II) ismeasured. Optionally, in order to make the signal stronger, it isacceptable to allow the bound structure (Ab I: NAIP) to be bound, atfirst, with secondary antibody that is not marker-labeled and then allowthe secondary antibody to be bound with the marker. Such bonding of thesecondary antibody with a marker-labeled molecular can be effected, forexample, by using the secondary antibody with biotin and tha marker withavidin. Further, it is also acceptable that an antibody (the tertiaryantibody) that recognizes a portion of the secondary antibody (e.g., Fcdomain) is marker-labeled, so that the tertiary antibody is bound withthe secondary antibody (II). The anti-NAIP monoclonal antibodies of thepresent invention may be used for both the primary antibody and thesecondary antibody. Alternatively, the anti-NAIP polyclonal antibody(the anti-serum of the animals immunized by the aforementionedpolypeptide, for example) may be used for one of the primary antibodyand the secondary antibody.

[0066] Although this second method can be carried out either in theliquid phase or on the solid phase, it is preferable to carry out themethod on the solid phase, in order to make the assay of extremely smallamounts and the operation as a whole easier. More specifically, thesolid phase method includes the steps of: providing the primary antibodyon a resin plate or the like so that the primary antibody isimmobilized; allowing the antibody on the solid state to be bound withNAIP; washing off the NAIP which is not bound to the antibody; allowingthe bound NAIP remaining on the plate to be bound with the secondaryantibody; and measuring the signal strength of the secondary antibody.This is what is called the “sandwich method”, and widely used as “ELISA”(enzyme linked immunospecific assay) when an enzyme is used as themarker.

[0067] In the methods described above, there is no particular limitationon the enzyme used as the marker, as long as the turn over number of theenzyme is relatively large, the enzyme is stable after being bound withthe antibody, the enzyme specifically acts on the substrate so that thesubstrate develops a color, and other required conditions are satisfied.Examples of the enzyme include the enzymes commonly used for EIA, suchas peroxydase, β-galactosidase, alkali-phosphatase, glucoseoxydase,acetylchorine-esterase, glucose-6-phosphorylation dehydrogenase, malicacid dehydrogenase and the like. Further, enzyme inhibitors andcoenzymes may also be used. Bonding of these enzymes with the monoclonalantibody can be carried out according to the known method which employsa cross-linking agent such as maleimide compounds. As the substrate, anysuitable known compounds may be used, depending on the types of theenzyme that is actually used. In a case in which peroxydase is used asthe enzyme, 3,3′5,5′-tetramethylbenzidine may be used as the substrate.In a case in which alkli-phosphatase is used as the enzyme,para-nitrophenol or the like may be used as the substrate.

[0068] In a case in which a radioactive isotope is used as the marker,examples of the radioactive isotope include those used in the standardRIA process such as ¹²⁵I and ³H. Examples of the fluorescent colorantsinclude those used in the standard fluorescent antibody method such asfluorescence isothiocyanate (FITC) or tetramethylrhodamineisothiocyanate (TRITC).

[0069] The assay kits of the present invention may be used for the“sandwich method” in which the aforementioned second assay method iscarried out on the solid phase. Such kits of various types arecommercially available in accordance with the types of the components tobe assayed. The assay kits of the present invention may be basicallyconstituted of various components used in known and commerciallyavailable kits, except that the aforementioned anti-NAIP monoclonalantibody and/or the anti-NAIP polyclonal antibody is used as theantibodies. In addition, the assay kits of the present inventionincluding the components described above may be provided with a washingsolution for washing off the NAIP which has not been bound and/or thesecondary antibody which has not been bound.

EXAMPLES

[0070] The present invention will be described in detail by exampleshereinafter. It should be noted, however, that the present invention isnot limited to any of these examples.

Example 1 Production of the Monoclonal Antibodies

[0071] (1) Preparation of the Immunogen

[0072] The 1056-2049th region of NAIP cDNA of which nucleotide sequenceis shown in SEQ ID NO: 2 was amplified, and the DNA fragment(NAIP.256-586) was inserted at the EcoR I site of pGEX-3X (by PharmaciaCo.). After confirming the nucleotide sequence, the host Escherichiacoli BL21 (DE3) pLysS was transformed by this recombinant vectorpGEX-3X(NAIP.256-586) and cultivated in the LB medium for 5 hours at 30°C. Thereafter, IPTG was added to the medium and the cultivation wascontinued at 20° C. for 3 hours. The bacteria was separated bycentrifuging, dissolved into the dissolving solution (PBS, TritonX-100), frozen at −80° C. and melted, and then subjected to ultrasonicdestruction. The product was centrifuged at 1000×g for 30 minutes, thesupernatant was introduced to a glutathione sepharose 4B column so as topass through it, whereby fusion protein GST-NAIP(256-586) was obtained.

[0073] In addition, the 2812-3447th region of NAIP cDNA of whichnucleotide sequence is shown in SEQ ID NO: 2 was amplified, and the DNAfragment (NAIP841-1052) was inserted at the BamHI-Sall site of pGEX-4X-3(by Pharmacia Co.). Thereafter, the same method as described above wasrepeated, thereby obtaining the fusion protein GST-NAIP(841-1052).

[0074] (2) Immunization of the Animal

[0075] 50 μg/μl of each of the fusion proteins obtained in theaforementioned (1) was dosed to a Balb/c mouse, intraperitoneally, asthe initial immunization. The second immunization was performed 2 weeksafter the initial immunization, and immunization was conducted totallysix times with one-week interval. At the initial immunication, thefusion protein was dosed in a state in which Freund complete adjuvant ofthe equal amount was mixed thereto. At the second to fifth immunization,the fusion protein was dosed in a state in which Freund incompleteadjuvant was mixed thereto. At the final immunization, only the fusionprotein solution was dosed.

[0076] (3) Fusion of Cells

[0077] The spleen cells were sterilely isolated three days after thefinal immunization. The collected spleen cells and the myeloma cell lineSP2/0-Ag14 derived from mice were mixed and then subjected to the fusingtreatment by using polyethylene glycol #4000. The obtained cells wereplanted on a 96-hole plate, and the fused cells were selected by the HATculture.

[0078] (4) Screening

[0079] An ELISA plate on which the NAIP polypeptide used as theimmunogen was provided on the solid state and an ELISA plate on whichGST was provided in the solid state were prepared. Clones that did notreact to the GST plate but reacted only to the NAIP plate were selectedand subjected to screening. Next, among the supernatants of the culturesof respective hybridomas, the wells reacted to the NAIP polypeptide wereregarded as positive. The cloning of the hybridomas was carried out byusing the positive wells in the limiting dilution method. The screeningprocess was repeated for the cultures of the hybridomas that weresupposed to have only single-type clones, whereby a plurality ofhybridomas was obtained. Among these plural hybridomas, hybridomas656-1, 656-2 and hnmc841 were deposited to National Institute ofBioscience and Human-Technology. The deposit Nos. of these hybridomasare FERM BP-6919 (hybridoma 656-1), FERM BP-6920 (hybridoma 656-2) andFERM BP-6921 (hybridoma hnmc841), respectively.

[0080] (5) Production of the Monoclonal Antibodies

[0081] Three types of the hybridomas obtained as described above weredosed to a Balb/c mice, intraperitoneally, and the ascites containingthe monoclonal antibody was collected after one week. From the collectedascites, the three types of monclonal antibodies hnmc365, hnmc381 andhnmc841 were purified by using an affinity column in which protein G wasused.

[0082] It was confirmed that the monoclonal antibody hnmc365, producedby hybridoma 656-1 which had been prepared by using fusion proteinGST-NAIP(256-586) as the immunogen, belongs to the subclass IgG1 and theepitope thereof is the amino acid sequence of the 254-368th region inSEQ ID NO: 1. It was also confirmed that the monoclonal antibody hnmc381produced by hybridoma 656-2 belongs to the subclass IgG2b and theepitope thereof is the amino acid sequence of the 373-387th region inSEQ ID NO: 1. Further, it was confirmed that the monoclonal antibodyhnmc841, produced by the hybridoma hnmc841 which had been prepared byusing fusion protein GST-NAIP(841-1052) as the immunogen, belongs to thesubclass IgG1 and the epitope thereof is the amino acid sequence of the841-1052nd region in SEQ ID NO: 1.

Example 2 Production of the Polyclonal Antibody

[0083] A rabbit (Japanese White Rabbit) was immunized by the standardmethod, by using as the immunogen the fusion protein GST-NAIP(256-586)prepared in a manner similar to that of Example 1 (1). The anti-serumwas then separated, and the polyclonal antibody was purified by asepharose 4B column in which the aforementioned fused proteins werebonded.

Example 3 Production of ELISA Kit

[0084] (1) Primary Antibody-Immobilized Plate

[0085] A solution (20 μg/ml) of the anti-NAIP monoclonal antibodyhnmc365 produced in Example 1 was dissolved into 10 mmol/l of potassiumphosphate buffer (pH 7.5) containing 150 mmol/l of sodium chloride and 1g/l of sodium azide. 50 μl of this solution was pipetted into each holeof a 96-hole plate for ELISA. The plate was stored at 4° C. for 16hours. Thereafter, the plate was washed with 10 mmol/l potassiumphosphate buffer (pH 7.5) containing 150 mmol/l sodium chloride, wherebythe plate on which the anti-NAIP monoclonal antibody was immobilized wasproduced.

[0086] (2) Biotinylated Secondary Antibody

[0087] 0.01 mmol of biotin-amidecaproic acid N-hydroxysuccinic imideester dissolved into N,N-dimethylformamide was added to 10 mg of theanti-NAIP polyclonal antibody produced in Example 2. The mixture wasstored at 25° C. for 3 hours and then subjected to dialysis for 16 hoursin 50 mmol/l potassium phosphate buffer (pH 7.4), whereby thebiotinylated anti-NAIP polyclonal antibody was produced.

[0088] (3) Marker to be Bound to the Secondary Antibody

[0089] A solution of horse radish peroxydase-labeled streptoavidin wasdiluted to the concentration of 0.5 μg/ml with 10 mmol/l potassiumphosphate buffer (pH 7.2) containing 150 mmol/l sodium chloride and 1g/L casein, whereby the marker solution was obtained.

Example 4 NAIP Assay

[0090] (1) Method of Operation

[0091] Sample solutions containing the purified NAIP at differentconcentrations were diluted with 10 mmol/l potassium phosphate buffer(pH 7.2) containing 150 mmol/l sodium chloride. 50 μl of each of thediluted solutions was pipetted into each hole of the plate on which theprimary antibodies had been provided in the solid state prepared inExample 3 (1). The plate was stored at 37° C. for 1 hour and then washedoff with 10 mmol/l potassium phosphate buffer (pH 7.2) containing 150mmol/l sodium chloride.

[0092] Next, the biotinated anti-NAIP polyclonal antibody of Example 3(2) was diluted to the concentration of 0.5 μg/ml with 10 mmol/lpotassium phosphate buffer (pH 7.2) containing 150 mmol/l sodiumchloride and 1 g/l casein. 100 μl of each of the diluted solutions waspipetted into each hole of the aforementioned plate. The plate wasstored at 37° C. for 1 hour and then washed off with 10 mmol/l potassiumphosphate buffer (pH 7.2) containing 150 mmol/l sodium chloride.

[0093] As the final step, 100 μl of the solution of horse radishperoxydase-labeled streptoavidin prepared in Example 3 (3) was pipettedinto each hole of the aforementioned plate. The plate was stored at 37°C. for 1 hour and then washed off with 10 mmol/l potassium phosphatebuffer (pH 7.2) containing 150 mmol/l sodium chloride.

[0094] (2) Color-Developing Reaction and Measurement of Absorbance

[0095] 3,3′,5,5′-tetramethylbenzidine was dissolved intoN,N-dimethylformamide so that the concentration of3,3′,5,5′-tetramethylbenzidine was 50 mmol/l. The obtained solution wasdiluted to {fraction (1/100)} with 100 mmol/l sodium accetate buffer (pH5.5) and then filtered by a filtering paper. 0.1 ml of aqueous hydrogenperoxide (10 g/l) was added to 10 ml of the solution, whereby the colordeveloping solution was obtained. 50 μl of the color developing solutionwas pipetted into each hole of the aforementioned plate. The plate wasstored at 30° C. for 30 minutes. Thereafter, 50 μl of sulfuric acid (2mol/l) was pipetted into each hole of the plate, so that the reactionstopped. Absorbance was then measured at 450 nm.

[0096] (3) Results

[0097]FIG. 1 is a graph that shows the relationship between theconcentration of the purified NAIP in the sample solution and theabsorbance measured by the aforementioned method. The concentration ofNAIP in the sample was measurable because the values thereof residedwithin the measurable range of 4 ng/ml to 20 ng/ml.

[0098] From the results, it was confirmed that, if the NAIPconcentration is unknown for a sample, the concentration of NAIP of thesample can be accurately assayed on the basis of the absorbance thereofby utilizing, for example, the measurement results as shown in FIG. 1 asthe reference line.

Example 5 Western Blot

[0099] (1) Preparation of Sample for SDS Gel Electrophoresis

[0100] Mononuclear cells were separated from 10 ml of normal humanperipheral blood by using Ficoll Paque PLUS (by Amasham-Pharmacia Co.).The obtained mononuclear cells were fixed by 5-10% trichloroacetic acidand then were separated by centrifuging. The separated cells weredissolved into a Tris buffer containing lithium dodesyl sulfate (2%),urea (8M), DTT (1%), and Triton X-100 (1%).

[0101] (2) Western Blot

[0102] Using the aforementioned sample carried out SDS gelelectrophoresis, and the result was transferred to a PVDF film. The PVDFfilm on which the transfer had been done was treated overnight at 4° C.with TBS containing skimmed milk (10%) and Tween 20 (0.05%). The PVDFfilm was then washed with TBS (TBST) containing Tween 20 (0.05%). Eachantibody was diluted with TBST in an appropriate manner and allowed toreact at the room temperature for 2 hours. Then, after washing withTBST, peroxydase-labeled anti-rabbit Ig antibody or anti-mouse Igantibody (by Amasham-Pharmacia Co.) was added for reaction thatproceeded at the room temperature for 1 hour. After washing with TBST,the treatment with the ECL PLUS reagent (by Amasham-Pharmacia Co.) andexposure onto an X-ray film followed, whereby signals were obtained.

[0103] (3) Results

[0104] The results are shown in FIG. 2. In all of the three types ofblots in which the monoclonal antibodies were used, signals of 160 kDawhich had been observed for the anti-NAIP polyclonal antibody weredetected.

[0105] From the aforementioned results, it was confirmed that themonoclonal antibodies hnmc365, hnmc381 and hnmc841 prepared in Example 1are monoclonal antibodies that specifically recognize NAIP and thusdetection of NAIP is possible by using these monoclonal antibodies.

Industrial Applicability

[0106] As described above in detail, the invention of the presentapplication allows simple but accurate assay of human apoptosisinhibitory protein (NAIP) present in a sample isolated from an organism.The present invention will facilitate better understanding of themechanism of patients developing symptoms of various apoptosis-relateddiseases, better diagnosis of the danger of developing the diseases,prevention of the development of the diseases or reduction of theseverity of the diseases, and development of the medical technique andmedicines for treatment.

1 2 1 1403 PRT Homo sapiens 1 Met Ala Thr Gln Gln Lys Ala Ser Asp GluArg Ile Ser Gln Phe Asp 1 5 10 15 His Asn Leu Leu Pro Glu Leu Ser AlaLeu Leu Gly Leu Asp Ala Val 20 25 30 Gln Leu Ala Lys Glu Leu Glu Glu GluGlu Gln Lys Glu Arg Ala Lys 35 40 45 Met Gln Lys Gly Tyr Asn Ser Gln MetArg Ser Glu Ala Lys Arg Leu 50 55 60 Lys Thr Phe Val Thr Tyr Glu Pro TyrSer Ser Trp Ile Pro Gln Glu 65 70 75 80 Met Ala Ala Ala Gly Phe Tyr PheThr Gly Val Lys Ser Gly Ile Gln 85 90 95 Cys Phe Cys Cys Ser Leu Ile LeuPhe Gly Ala Gly Leu Thr Arg Leu 100 105 110 Pro Ile Glu Asp His Lys ArgPhe His Pro Asp Cys Gly Phe Leu Leu 115 120 125 Asn Lys Asp Val Gly AsnIle Ala Lys Tyr Asp Ile Arg Val Lys Asn 130 135 140 Leu Lys Ser Arg LeuArg Gly Gly Lys Met Arg Tyr Gln Glu Glu Glu 145 150 155 160 Ala Arg LeuAla Ser Phe Arg Asn Trp Pro Phe Tyr Val Gln Gly Ile 165 170 175 Ser ProCys Val Leu Ser Glu Ala Gly Phe Val Phe Thr Gly Lys Gln 180 185 190 AspThr Val Gln Cys Phe Ser Cys Gly Gly Cys Leu Gly Asn Trp Glu 195 200 205Glu Gly Asp Asp Pro Trp Lys Glu His Ala Lys Trp Phe Pro Lys Cys 210 215220 Glu Phe Leu Arg Ser Lys Lys Ser Ser Glu Glu Ile Thr Gln Tyr Ile 225230 235 240 Gln Ser Tyr Lys Gly Phe Val Asp Ile Thr Gly Glu His Phe ValAsn 245 250 255 Ser Trp Val Gln Arg Glu Leu Pro Met Ala Ser Ala Tyr CysAsn Asp 260 265 270 Ser Ile Phe Ala Tyr Glu Glu Leu Arg Leu Asp Ser PheLys Asp Trp 275 280 285 Pro Arg Glu Ser Ala Val Gly Val Ala Ala Leu AlaLys Ala Gly Leu 290 295 300 Phe Tyr Thr Gly Ile Lys Asp Ile Val Gln CysPhe Ser Cys Gly Gly 305 310 315 320 Cys Leu Glu Lys Trp Gln Glu Gly AspAsp Pro Leu Asp Asp His Thr 325 330 335 Arg Cys Phe Pro Asn Cys Pro PheLeu Gln Asn Met Lys Ser Ser Ala 340 345 350 Glu Val Thr Pro Asp Leu GlnSer Arg Gly Glu Leu Cys Glu Leu Leu 355 360 365 Glu Thr Thr Ser Glu SerAsn Leu Glu Asp Ser Ile Ala Val Gly Pro 370 375 380 Ile Val Pro Glu MetAla Gln Gly Glu Ala Gln Trp Phe Gln Glu Ala 385 390 395 400 Lys Asn LeuAsn Glu Gln Leu Arg Ala Ala Tyr Thr Ser Ala Ser Phe 405 410 415 Arg HisMet Ser Leu Leu Asp Ile Ser Ser Asp Leu Ala Thr Asp His 420 425 430 LeuLeu Gly Cys Asp Leu Ser Ile Ala Ser Lys His Ile Ser Lys Pro 435 440 445Val Gln Glu Pro Leu Val Leu Pro Glu Val Phe Gly Asn Leu Asn Ser 450 455460 Val Met Cys Val Glu Gly Glu Ala Gly Ser Gly Lys Thr Val Leu Leu 465470 475 480 Lys Lys Ile Ala Phe Leu Trp Ala Ser Gly Cys Cys Pro Leu LeuAsn 485 490 495 Arg Phe Gln Leu Val Phe Tyr Leu Ser Leu Ser Ser Thr ArgPro Asp 500 505 510 Glu Gly Leu Ala Ser Ile Ile Cys Asp Gln Leu Leu GluLys Glu Gly 515 520 525 Ser Val Thr Glu Met Cys Met Arg Asn Ile Ile GlnGln Leu Lys Asn 530 535 540 Gln Val Leu Phe Leu Leu Asp Asp Tyr Lys GluIle Cys Ser Ile Pro 545 550 555 560 Gln Val Ile Gly Lys Leu Ile Gln LysAsn His Leu Ser Arg Thr Cys 565 570 575 Leu Leu Ile Ala Val Arg Thr AsnArg Ala Arg Asp Ile Arg Arg Tyr 580 585 590 Leu Glu Thr Ile Leu Glu IleLys Ala Phe Pro Phe Tyr Asn Thr Val 595 600 605 Cys Ile Leu Arg Lys LeuPhe Ser His Asn Met Thr Arg Leu Arg Lys 610 615 620 Phe Met Val Tyr PheGly Lys Asn Gln Ser Leu Gln Lys Ile Gln Lys 625 630 635 640 Thr Pro LeuPhe Val Ala Ala Ile Cys Ala His Trp Phe Gln Tyr Pro 645 650 655 Phe AspPro Ser Phe Asp Asp Val Ala Val Phe Lys Ser Tyr Met Glu 660 665 670 ArgLeu Ser Leu Arg Asn Lys Ala Thr Ala Glu Ile Leu Lys Ala Thr 675 680 685Val Ser Ser Cys Gly Glu Leu Ala Leu Lys Gly Phe Phe Ser Cys Cys 690 695700 Phe Glu Phe Asn Asp Asp Asp Leu Ala Glu Ala Gly Val Asp Glu Asp 705710 715 720 Glu Asp Leu Thr Met Cys Leu Met Ser Lys Phe Thr Ala Gln ArgLeu 725 730 735 Arg Pro Phe Tyr Arg Phe Leu Ser Pro Ala Phe Gln Glu PheLeu Ala 740 745 750 Gly Met Arg Leu Ile Glu Leu Leu Asp Ser Asp Arg GlnGlu His Gln 755 760 765 Asp Leu Gly Leu Tyr His Leu Lys Gln Ile Asn SerPro Met Met Thr 770 775 780 Val Ser Ala Tyr Asn Asn Phe Leu Asn Tyr ValSer Ser Leu Pro Ser 785 790 795 800 Thr Lys Ala Gly Pro Lys Ile Val SerHis Leu Leu His Leu Val Asp 805 810 815 Asn Lys Glu Ser Leu Glu Asn IleSer Glu Asn Asp Asp Tyr Leu Lys 820 825 830 His Gln Pro Glu Ile Ser LeuGln Met Gln Leu Leu Arg Gly Leu Trp 835 840 845 Gln Ile Cys Pro Gln AlaTyr Phe Ser Met Val Ser Glu His Leu Leu 850 855 860 Val Leu Ala Leu LysThr Ala Tyr Gln Ser Asn Thr Val Ala Ala Cys 865 870 875 880 Ser Pro PheVal Leu Gln Phe Leu Gln Gly Arg Thr Leu Thr Leu Gly 885 890 895 Ala LeuAsn Leu Gln Tyr Phe Phe Asp His Pro Glu Ser Leu Ser Leu 900 905 910 LeuArg Ser Ile His Phe Pro Ile Arg Gly Asn Lys Thr Ser Pro Arg 915 920 925Ala His Phe Ser Val Leu Glu Thr Cys Phe Asp Lys Ser Gln Val Pro 930 935940 Thr Ile Asp Gln Asp Tyr Ala Ser Ala Phe Glu Pro Met Asn Glu Trp 945950 955 960 Glu Arg Asn Leu Ala Glu Lys Glu Asp Asn Val Lys Ser Tyr MetAsp 965 970 975 Met Gln Arg Arg Ala Ser Pro Asp Leu Ser Thr Gly Tyr TrpLys Leu 980 985 990 Ser Pro Lys Gln Tyr Lys Ile Pro Cys Leu Glu Val AspVal Asn Asp 995 1000 1005 Ile Asp Val Val Gly Gln Asp Met Leu Glu IleLeu Met Thr Val Phe 1010 1015 1020 Ser Ala Ser Gln Arg Ile Glu Leu HisLeu Asn His Ser Arg Gly Phe 1025 1030 1035 1040 Ile Glu Ser Ile Arg ProAla Leu Glu Leu Ser Lys Ala Ser Val Thr 1045 1050 1055 Lys Cys Ser IleSer Lys Leu Glu Leu Ser Ala Ala Glu Gln Glu Leu 1060 1065 1070 Leu LeuThr Leu Pro Ser Leu Glu Ser Leu Glu Val Ser Gly Thr Ile 1075 1080 1085Gln Ser Gln Asp Gln Ile Phe Pro Asn Leu Asp Lys Phe Leu Cys Leu 10901095 1100 Lys Glu Leu Ser Val Asp Leu Glu Gly Asn Ile Asn Val Phe SerVal 1105 1110 1115 1120 Ile Pro Glu Glu Phe Pro Asn Phe His His Met GluLys Leu Leu Ile 1125 1130 1135 Gln Ile Ser Ala Glu Tyr Asp Pro Ser LysLeu Val Lys Leu Ile Gln 1140 1145 1150 Asn Ser Pro Asn Leu His Val PheHis Leu Lys Cys Asn Phe Phe Ser 1155 1160 1165 Asp Phe Gly Ser Leu MetThr Met Leu Val Ser Cys Lys Lys Leu Thr 1170 1175 1180 Glu Ile Lys PheSer Asp Ser Phe Phe Gln Ala Val Pro Phe Val Ala 1185 1190 1195 1200 SerLeu Pro Asn Phe Ile Ser Leu Lys Ile Leu Asn Leu Glu Gly Gln 1205 12101215 Gln Phe Pro Asp Glu Glu Thr Ser Glu Lys Phe Ala Tyr Ile Leu Gly1220 1225 1230 Ser Leu Ser Asn Leu Glu Glu Leu Ile Leu Pro Thr Gly AspGly Ile 1235 1240 1245 Tyr Arg Val Ala Lys Leu Ile Ile Gln Gln Cys GlnGln Leu His Cys 1250 1255 1260 Leu Arg Val Leu Ser Phe Phe Lys Thr LeuAsn Asp Asp Ser Val Val 1265 1270 1275 1280 Glu Ile Ala Lys Val Ala IleSer Gly Gly Phe Gln Lys Leu Glu Asn 1285 1290 1295 Leu Lys Leu Ser IleAsn His Lys Ile Thr Glu Glu Gly Tyr Arg Asn 1300 1305 1310 Phe Phe GlnAla Leu Asp Asn Met Pro Asn Leu Gln Glu Leu Asp Ile 1315 1320 1325 SerArg His Phe Thr Glu Cys Ile Lys Ala Gln Ala Thr Thr Val Lys 1330 13351340 Ser Leu Ser Gln Cys Val Leu Arg Leu Pro Arg Leu Ile Arg Leu Asn1345 1350 1355 1360 Met Leu Ser Trp Leu Leu Asp Ala Asp Asp Ile Ala LeuLeu Asn Val 1365 1370 1375 Met Lys Glu Arg His Pro Gln Ser Lys Tyr LeuThr Ile Leu Gln Lys 1380 1385 1390 Trp Ile Leu Pro Phe Ser Pro Ile IleGln Lys 1395 1400 1403 2 5984 DNA Homo sapiens CDC (292)..(4500) 2acaaaaggtc ctgtgctcac ctgggaccct tctggacgtt gccctgtgtt cctcttcgcc 60tgcctgttca tctacgacga accccgggta ttgaccccag acaacaatgc cacttcatat 120tggggacttc gtctgggatt ccaaggtgca ttcattgcaa agttccttaa atattttctc 180actgcttcct actaaaggac ggacagagca tttgttcttc agccacatac tttccttcca 240ctggccagca ttctcctcta ttagactaga actgtggata aacctcagaa aatggccacc 300cagcagaaag cctctgacga gaggatctcc cagtttgatc acaatttgct gccagagctg 360tctgctcttc tgggcctaga tgcagttcag ttggcaaagg aactagaaga agaggagcag 420aaggagcgag caaaaatgca gaaaggctac aactctcaaa tgcgcagtga agcaaaaagg 480ttaaagactt ttgtgactta tgagccgtac agctcatgga taccacagga gatggcggcc 540gctgggtttt acttcactgg ggtaaaatct gggattcagt gcttctgctg tagcctaatc 600ctctttggtg ccggcctcac gagactcccc atagaagacc acaagaggtt tcatccagat 660tgtgggttcc ttttgaacaa ggatgttggt aacattgcca agtacgacat aagggtgaag 720aatctgaaga gcaggctgag aggaggtaaa atgaggtacc aagaagagga ggctagactt 780gcatccttca ggaactggcc attttatgtc caagggatat ccccttgtgt gctctcagag 840gctggctttg tctttacagg taaacaggac acggtacagt gtttttcctg tggtggatgt 900ttaggaaatt gggaagaagg agatgatcct tggaaggaac atgccaaatg gttccccaaa 960tgtgaatttc ttcggagtaa gaaatcctca gaggaaatta cccagtatat tcaaagctac 1020aagggatttg ttgacataac gggagaacat tttgtgaatt cctgggtcca gagagaatta 1080cctatggcat cagcttattg caatgacagc atctttgctt acgaagaact acggctggac 1140tcttttaagg actggccccg ggaatcagct gtgggagttg cagcactggc caaagcaggt 1200cttttctaca caggtataaa ggacatcgtc cagtgctttt cctgtggagg gtgtttagag 1260aaatggcagg aaggtgatga cccattagac gatcacacca gatgttttcc caattgtcca 1320tttctccaaa atatgaagtc ctctgcggaa gtgactccag accttcagag ccgtggtgaa 1380ctttgtgaat tactggaaac cacaagtgaa agcaatcttg aagattcaat agcagttggt 1440cctatagtgc cagaaatggc acagggtgaa gcccagtggt ttcaagaggc aaagaatctg 1500aatgagcagc tgagagcagc ttataccagc gccagtttcc gccacatgtc tttgcttgat 1560atctcttccg atctggccac ggaccacttg ctgggctgtg atctgtctat tgcttcaaaa 1620cacatcagca aacctgtgca agaacctctg gtgctgcctg aggtctttgg caacttgaac 1680tctgtcatgt gtgtggaggg tgaagctgga agtggaaaga cggtcctcct gaagaaaata 1740gcttttctgt gggcatctgg atgctgtccc ctgttaaaca ggttccagct ggttttctac 1800ctctccctta gttccaccag accagacgag gggctggcca gtatcatctg tgaccagctc 1860ctagagaaag aaggatctgt tactgaaatg tgcatgagga acattatcca gcagttaaag 1920aatcaggtct tattcctttt agatgactac aaagaaatat gttcaatccc tcaagtcata 1980ggaaaactga ttcaaaaaaa ccacttatcc cggacctgcc tattgattgc tgtccgtaca 2040aacagggcca gggacatccg ccgataccta gagaccattc tagagatcaa agcatttccc 2100ttttataata ctgtctgtat attacggaag ctcttttcac ataatatgac tcgtctgcga 2160aagtttatgg tttactttgg aaagaaccaa agtttgcaga agatacagaa aactcctctc 2220tttgtggcgg cgatctgtgc tcattggttt cagtatcctt ttgacccatc ctttgatgat 2280gtggctgttt tcaagtccta tatggaacgc ctttccttaa ggaacaaagc gacagctgaa 2340attctcaaag caactgtgtc ctcctgtggt gagctggcct tgaaagggtt tttttcatgt 2400tgctttgagt ttaatgatga tgatctcgca gaagcagggg ttgatgaaga tgaagatcta 2460accatgtgct tgatgagcaa atttacagcc cagagactaa gaccattcta ccggttttta 2520agtcctgcct tccaagaatt tcttgcgggg atgaggctga ttgaactcct ggattcagat 2580aggcaggaac atcaagattt gggactgtat catttgaaac aaatcaactc acccatgatg 2640actgtaagcg cctacaacaa ttttttgaac tatgtctcca gcctcccttc aacaaaagca 2700gggcccaaaa ttgtgtctca tttgctccat ttagtggata acaaagagtc attggagaat 2760atatctgaaa atgatgacta cttaaagcac cagccagaaa tttcactgca gatgcagtta 2820cttaggggat tgtggcaaat ttgtccacaa gcttactttt caatggtttc agaacattta 2880ctggttcttg ccctgaaaac tgcttatcaa agcaacactg ttgctgcgtg ttctccattt 2940gttttgcaat tccttcaagg gagaacactg actttgggtg cgcttaactt acagtacttt 3000ttcgaccacc cagaaagctt gtcattgttg aggagcatcc acttcccaat acgaggaaat 3060aagacatcac ccagagcaca tttttcagtt ctggaaacat gttttgacaa atcacaggtg 3120ccaactatag atcaggacta tgcttctgcc tttgaaccta tgaatgaatg ggagcgaaat 3180ttagctgaaa aagaggataa tgtaaagagc tatatggata tgcagcgcag ggcatcacca 3240gaccttagta ctggctattg gaaactttct ccaaagcagt acaagattcc ctgtctagaa 3300gtcgatgtga atgatattga tgttgtaggc caggatatgc ttgagattct aatgacagtt 3360ttctcagctt cacagcgcat cgaactccat ttaaaccaca gcagaggctt tatagaaagc 3420atccgcccag ctcttgagct gtctaaggcc tctgtcacca agtgctccat aagcaagttg 3480gaactcagcg cagccgaaca ggaactgctt ctcaccctgc cttccctgga atctcttgaa 3540gtctcaggga caatccagtc acaagaccaa atctttccta atctggataa gttcctgtgc 3600ctgaaagaac tgtctgtgga tctggagggc aatataaatg ttttttcagt cattcctgaa 3660gaatttccaa acttccacca tatggagaaa ttattgatcc aaatttcagc tgagtatgat 3720ccttccaaac tagtaaaatt aattcaaaat tctccaaacc ttcatgtttt ccatctgaag 3780tgtaacttct tttcggattt tgggtctctc atgactatgc ttgtttcctg taagaaactc 3840acagaaatta agttttcgga ttcatttttt caagccgtcc catttgttgc cagtttgcca 3900aattttattt ctctgaagat attaaatctt gaaggccagc aatttcctga tgaggaaaca 3960tcagaaaaat ttgcctacat tttaggttct cttagtaacc tggaagaatt gatccttcct 4020actggggatg gaatttatcg agtggccaaa ctgatcatcc agcagtgtca gcagcttcat 4080tgtctccgag tcctctcatt tttcaagact ttgaatgatg acagcgtggt ggaaattgcc 4140aaagtagcaa tcagtggagg tttccagaaa cttgagaacc taaagctttc aatcaatcac 4200aagattacag aggaaggata cagaaatttc tttcaagcac tggacaacat gccaaacttg 4260caggagttgg acatctccag gcatttcaca gagtgtatca aagctcaggc cacaacagtc 4320aagtctttga gtcaatgtgt gttacgacta ccaaggctca ttagactgaa catgttaagt 4380tggctcttgg atgcagatga tattgcattg cttaatgtca tgaaagaaag acatcctcaa 4440tctaagtact taactattct ccagaaatgg atactgccgt tctctccaat cattcagaaa 4500taaaagattc agctaaaaac tgctgaatca ataatttgtc ttggggcata ttgaggatgt 4560aaaaaaagtt gttgattaat gctaaaaacc aaattatcca aaattatttt attaaatatt 4620gcatacaaaa gaaaatgtgt aaggcttgct aaaaaacaaa acaaaacaaa acacagtcct 4680gcatactcac caccaagctc aagaaataaa tcatcaccaa tacctttgag gtccctgagt 4740aatccacccc agctaaaggc aaacccttca atcaagttta tacagcaaac cctccattgt 4800ccatggtcaa cagggaaggg gttggggaca ggtctgccaa tctatctaaa agccacaata 4860tggaagaagt attcaattta tataataaat ggctaactta acggttgaat cactttcata 4920catggatgaa acgggtttaa cacaggatcc acatgaatct tctgtgggcc aaaatatgtt 4980ccttaatcct tgtagaacct gtcttctata ttgaactagc tttggtacag tagagttaac 5040ttactttcca tttatccact gccaatataa agaggaaaca ggggttaggg aaaaatgact 5100tcattccaga ggcttctcag agttcaacat atgctataat ttagaatttt cttatgaatc 5160cactctactt gggtagaaaa tattttatct ctagtgattg catattattt ccatatcata 5220gtatttcata gtattatatt tgatatgagt gtctatatca atgtcagtgt ccagaatttc 5280gttcctacca gttgagtagt tttctgaacg gccagaagac cattcgaaat tcatgatact 5340actataagtt ggtaaacaac catactttta tcctcatttt tattctcact aagaaaaaag 5400tcaactcccc tccccttgcc caagtatgaa atatagggac agtatgtatg gtgtggtctc 5460atttgtttag aaaaccactt atgactgggt gcggtggctc acacctgtaa tcccagcact 5520ttgggaggct gaggcgggcg aatcatttga ggtgaggagt tcgagaccgg cctggccagc 5580atggtgaaac cccatttttg ctaaaggtac aaaaattagc caggtgtggt ggcacatgcc 5640tgtggtccca gccactgggg cggctgagac gcaggacttg cttgaacccg ggaggcagag 5700gttgcagtga gccgagatgg cgccactgca ttccagcctg ggcaacagag caagaccctg 5760tctgtttcaa aacaaaaaac aaaaccactt atattgctag ctacattaag aatttctgaa 5820tatgttactg agcttgcttg tggtaaccat ttataatatc agaaagtata tgtacaccaa 5880aacatgttga acatccatgt tgtacaactg aaatataaat aattttgtca attataccta 5940aataaaactg gaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaa 5984

1. An anti-NAIP monoclonal antibody recognizing a human apoptosisinhibitory protein NAIP having the amino acid sequence of SEQ ID NO: 1,which is produced from hybridoma prepared by fusing a myeloma cell linewith antibody-producing cell of mammal immunized by antigen containing apolypeptide which comprises amino acid sequence of the 256-586th, the841-1052nd or parts thereof in SEQ ID NO: 1
 2. Anti-NAIP monoclonalantibody hnmc365, which is produced from hybridoma 656-1 (FERM BP-6919),and its epitope is the 354-365th region in SEQ ID NO:
 1. 3. Anti-NAIPmonoclonal antibody hnmc381, which is produced from hybridoma 656-2(FERM BP-6920), and its epitope is the 373-387th region in SEQ ID NO: 1.4. Anti-NAIP monoclonal antibody hnmc841, which is produced fromhybridoma 841 (FERM BP-6921), and its epitope is the 841-1052nd regionin SEQ ID NO:1.
 5. A method of assaying NAIP, which comprises contactinga marker-labeled anti-NAIP monoclonal antibody of claim 1 with a samplecontaining NAIP thereby binding the marker-labeled antibody with NAIP,and measuring signal strength of the marker in the bound structure. 6.The method of assaying NAIP of claim 5, wherein the anti-NAIP monoclonalantibody is any one of the monoclonal antibodies of claims 2 to
 4. 7.The method of assaying NAIP of claim 5 or 6, wherein the marker is anenzyme, a radioactive isotope or a fluorescent colorant.
 8. A method ofassaying NAIP which comprises contacting an anti-NAIP primary antibodywith a sample containing NAIP thereby binding the primary antibody withNAIP, further binding the bound structure with an anti-NAIP secondaryantibody, and measuring signal strength of a marker bound with thesecondary antibody, wherein: (1) the primary antibody and the secondaryantibody are both the anti-NAIP monoclonal antibody of claim 1; (2) theprimary antibody is the anti-NAIP monoclonal antibody of claim 1 and thesecondary antibody is an anti-NAIP polyclonal antibody; or (3) theprimary antibody is an anti-NAIP polyclonal antibody and the secondaryantibody is the anti-NAIP monoclonal antibody of claim
 1. 9. The methodof assaying NAIP of claim 8, wherein the primary antibody is immobilizedon solid phase.
 10. The method of assaying NAIP of claim 8 or 9, whereinthe anti-NAIP monoclonal antibody is any one of the monoclonalantibodies of claims 2 to
 4. 11. The method of assaying NAIP of claim 8,9 or 10, wherein the marker is an enzyme, a radioactive isotope or afluorescent colorant.
 12. A NAIP assay kit at least including: (a) aplate on which an anti-NAIP primary antibody is immobilized; and (b) ananti-NAIP secondary antibody labeled with a marker, wherein: (1) theprimary antibody and the secondary antibody are both the anti-NAIPmonoclonal antibody of claim 1; (2) the primary antibody is theanti-NAIP monoclonal antibody of claim 1 and the secondary antibody isan anti-NAIP polyclonal antibody; or (3) the primary antibody is ananti-NAIP polyclonal antibody and the secondary antibody is theanti-NAIP monoclonal antibody of claim
 1. 13 The NAIP assay kit of claim12, wherein the anti-NAIP monoclonal antibody is any one of themonoclonal antibodies of claims 2 to
 4. 14. The NAIP assay kit of claim12 or 13, wherein the marker is a radioactive isotope or a fluorescentcolorant.
 15. The NAIP assaying kit of claim 12 or 13, wherein themarker is an enzyme and the kit further includes: (c) a substrate whichdevelops a color by the enzyme activity.
 16. A NAIP assaying kit atleast including: (a) a plate on which an anti-NAIP primary antibody isimmobilized; (b) an anti-NAIP secondary antibody; and (c) a marker to bebound with the secondary antibody, wherein: (1) the primary antibody andthe secondary antibody are both the anti-NAIP monoclonal antibody ofclaim 1; (2) the primary antibody is the anti-NAIP monoclonal antibodyof claim 1 and the secondary antibody is an anti-NAIP polyclonalantibody; or (3) the primary antibody is an anti-NAIP polyclonalantibody and the secondary antibody is the anti-NAIP monoclonal antibodyof claim
 1. 17. The NAIP assaying kit of claim 16, wherein the anti-NAIPmonoclonal antibody is any one of the monoclonal antibodies of claims 2to
 4. 18. The NAIP assaying kit of claim 16 or 17, wherein the marker isa radioactive isotope or a fluorescent colorant.
 19. The NAIP assayingkit of claim 16 or 17, wherein the marker is an enzyme and the kitfurther includes: (d) a substrate which develops a color by the enzymeactivity.